Elaphoglossum is a taxonomically challenging fern genus with about 600 species. The present study concerns a group of five Elaphoglossum species mainly distributed in Mexico, four of which were recovered as monophyletic and are here called the Mexican-petiolatum clade (E. petiolatum, E. potosianum, E. pringlei, and E. rzedowskii). The fifth species, E. muelleri, is distributed in Central America and Mexico and is morphologically similar to species of the Mexican-petiolatum clade. This study investigates the spores of these five species and has three objectives: first, to describe and compare their spore ornamentation; second, to analyze the variation in spore size among the species and within the widely distributed species E. petiolatum; and third, to assess whether perine ornamentation and exine size are useful to recognize species of the Mexican-petiolatum clade and/or for species delimitation. We studied 26 herbarium specimens of the five species and used scanning electron microscopy and statistical analyses to examine, measure, and analyze 133 spores. All the spores examined have uniform primary ornamentation (broad and discontinuous folds), but the secondary ornamentation is variable. Spores from Mexican specimens, regardless of their species, have small spines with narrow bases; spores of E. petiolatum from Central America have spines with wider bases; and spores of E. petiolatum from the West Indies have verrucae. Statistical analyses revealed that there are significant differences in spore size among all the studied species and within specimens identified as E. petiolatum. Within E. petiolatum, spores from the Dominican Republic are larger than those from the other countries. The results support our hypothesis that E. muelleri is part of the Mexican-petiolatum clade and suggest that E. petiolatum in Mexico, Central America, and the West Indies likely represent different taxa. Spore size differences help distinguish closely related species in the group such as E. pringlei and E. rzedowskii and indicate that there might be polyploid species within the Mexican-petiolatum clade.